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. 2012 May 8;10(5):e1001326. doi: 10.1371/journal.pbio.1001326

Figure 5. NOX supports glycolysis in cells with mitochondrial respiratory defects induced by POLGdn.

Figure 5

(A) Inhibition of glucose uptake by siRNA knockdown of NOX1 or p22phox in cells with mitochondrial respiratory defects (Tet/on, day 8), but not in cells with intact mitochondria (Tet/off). p<0.05 (n = 3). Insert: Tet/off and Tet/on (at day 4) cells were transiently transfected with p22phox siRNA and the knockdown efficiency was detected by anti-p22phox antibody using Western blot. Non-targeting control siRNA (Scram or sc) was used as negative control. (B) Effect of NOX1 knockdown on ATP contents in cells with intact mitochondria (Tet/off) and cells with mitochondrial respiratory defect (Tet/on, day 8). p<0.01 (n = 4). (C) HPLC analysis of cellular NAD+ and NADH levels. Standard NAD+ and NADH were used as references, which were monitored simultaneously at 260 nm and 340 nm, respectively. A lower NAD+ content was detected in the Tet/on cells with mitochondrial respiratory dysfunction and higher glycolytic activity that consumed more of NAD+. Inhibition of NOX activity by p22phox siRNA and DPI resulted in further decrease in cellular NAD+ level. (D) Quantitation of intracellular NAD+ and NADH in triplicate experiments, using HPLC method as described above. (E) Effect of POLGdn expression on cellular NADP+/NADPH ratio. *** p<0.001. (F) Knockdown of p22phox decreased NADPH/NADH oxidase activity. Comparing to the control siRNA knockdown in Tet/on (d8) cells, p22phox knockdown significantly decreased cellular NADPH/NADH oxidase activity using either NADHP or NADH as substrate. All error bars, ±SD. ** p<0.01; *** p<0.001 and n = 3. (G) HIF-1α is not stabilized following POLGdn induction. Protein level of HIF-1α in cells at the indicated time points after POLGdn induction was assayed by Western blot analysis. Tet/off cells with 2% oxygen were used as positive control. β-actin was used as a loading control.